A peptide-binding motif for I-A(g7), the class II major histocompatibility complex (MHC) molecule of NOD and Biozzi AB/H mice

The class II major histocompatibility complex molecule I-A(g7) is strongly linked to the development of spontaneous insulin-dependent diabetes mellitus (IDDM) in non obese diabetic mice and to the induction of experimental allergic encephalomyelitis in Biozzi AB/H mice. Structurally, it resembles the HLA-DQ molecules associated with human IDDM, in having a non-Asp residue at position 57 in its beta chain. To identify the requirements for peptide binding to I-A(g7) and thereby potentially pathogenic T cell epitopes, we analyzed a known I-A(g7)-restricted T cell epitope, hen egg white lysozyme (HEL) amino acids 9-27. NH2- and COOH-terminal truncations demonstrated that the minimal epitope for activation of the T cell hybridoma 2D12.1 was M12-R21 and the minimum sequence for direct binding to purified I-A(g7) M12-Y20/K13-R21. Alanine (A) scanning revealed two primary anchors for binding at relative positions (p) 6 (L) and 9 (Y) in the HEL epitope. The critical role of both anchors was demonstrated by incorporating L and Y in poly(A) backbones at the same relative positions as in the HEL epitope. Well-tolerated, weakly tolerated, and nontolerated residues were identified by analyzing the binding of peptides containing multiple substitutions at individual positions. Optimally, p6 was a large, hydrophobic residue (L, I, V, M), whereas p9 was aromatic and hydrophobic (Y or F) or positively charged (K, R). Specific residues were not tolerated at these and some other positions. A motif for binding to I-A(g7) deduced from analysis of the model HEL epitope was present in 27/30 (90%) of peptides reported to be I-A(g7)-restricted T cell epitopes or eluted from I-A(g7). Scanning a set of overlapping peptides encompassing human proinsulin revealed the motif in 6/6 good binders (sensitivity = 100%) and 4/13 weak or non-binders (specificity = 70%). This motif should facilitate identification of autoantigenic epitopes relevant to the pathogenesis and immunotherapy of IDDM.

The subfamily Aephnidiogeninae Yamaguti, 1934 (Digenea: Lepocreadiidae), its status and that of the genera Aephnidiogenes Nicoll, 1915, Holorchis Stossich, 1901, Austroholorchis n g, Pseudaephnidiogenes Yamaguti, 1971, Pseudoholorchis Yamaguti, 1958 and N

The status of all of the putative member genera of the subfamily Aephnidiogeninae is reconsidered, based mainly on the morphology of the terminal genitalia, Aephnidiogenes Nicoll, 1915 is the only genus retained in the Aaephnidiogeninae. Aephnidiogenes major Yamaguti, 1934 from Diagramma labiosum from the southern Great Barrier Reef is redescribed with particular reference to the terminal genitalia, and is shown to lack a true cirrussac, a condition considered to be diagnostic of the Aephnidiogeninae. Holorchis Stossich, 1901 is placed in the subfamily Lepidapedinae. Holorchis pycnoporus Stossich, 1901 from Pagellus acarne from off Spanish Sahara and from Diplodus vulgaris from off Italy and H. legendrei Dollfus, 1946 from Sparodon durbanensis and D. sargus from off eastern Cape Province, South Africa and from Pagellus erythrinus from the Adriatic Sea and Italy are studied and illustrated. The terminal genitalia of H. pycnoporus are found to be enigmatic, but those of H. legendrei are found to fit clearly into the 'Lepidapedon-like' pattern. A new genus Austroholorchis is erected in the Lepidapedinae, with A. sprenti (Gibson, 1987) n. comb. as the type-species. Its diagnostic features are its ani, infundibuliform oral sucker and the position of the ovary at about mid-level of the uterus. A. sprenti is illustrated, its hosts in Queensland waters being Sillago maculata, S, analis and S. ciliata. A, levis n. sp. is described from Sillago bassensis from south-western Western Australia. The genus Pseudaephnidiogenes Yamaguti, 1971 is placed in the Lepidapedinae. P. rhabdosargi (Prudhoe, 1956) from Rhabdosargus sarba from off Natal, South Africa is illustrated and the terminal genitalia of P. rhabdosargi from R. sarba and from R. holubi from off eastern Cape Province and Pseudaephnidiogenes vossi Bray, 1985 from Caffrogobius nudiceps from off eastern Cape Province, South Africa are illustrated. The genus Pseudoholorchis Yamaguti, 1958 is placed in the subfamily Lepocreadiinae. The terminal genitalia of P. pulcher (Manter, 1954) from Latridopsis ciliaris from New Zealand are illustrated, The genus Neolepocreadium Thomas, 1960 is placed in the Lepocreadiidae.

Postlepidapedon Zdzitowiecki, 1993 and Gibsonivermis n g (Digenea: Lepocreadiidae) from fishes of the southern Great Barrier Reef, Australia, and their relationship to Intusatrium Durio & Manter, 1968

The genus Intusatrium Durio & Manter, 1968 is redefined based on a re-examination of paratypes of the type-species, I. robustum Durio & Manter, 1968, and is considered monotypic with characteristic terminal genitalia: internal seminal vesicle elongate tubular, with rather thick wall, divided by slight change in wall thickness into longer proximal and shorter distal region; pars prostatica subcylindrical; ejaculatory duct relatively short, with wrinkled/wall. The genus Postlepidapedon Zdzitowiecki, 1993 is redefined and Intusatrium secundum Durio & Manter, 1968 is attributed to it as a new combination. Postlepidapedon secundum n. comb. is redescribed from a paratype and new material from Choerodon graphicus. P. spissum n. sp. from Choerodon venustus, C. cyanodus, C. fasciatus and C. schoenleinii is recognised on the basis of its thick-walled internal seminal vesicle. I! uberis n. sp. from Choerodon schoenleinii and C. venustus is distinguished by the shape and contents of the cirrus-sac with narrow, convoluted internal seminal vesicle, large vesicular pars prostatica and short, muscular ejaculatory duct. A new genus, Gibsonivermis, erected for Intusatrium berryi Gibson, 1987, is characterised by the elongate narrow cirrus-sac and a uroproct. G. berryi n. comb. is redescribed from Sillago ciliata, S. maculata and Sillago sp.

Crystal structure of Escherichia coli xanthine phosphoribosyltransferase

Xanthine phosphoribosyltransferase (XPRT; EC 2.4.2.22) from Escherichia coil is a tetrameric enzyme having 152 residues per subunit. XPRT catalyzes the transfer of the phosphoribosyl group from 5-phospho-alpha-D-ribosyl l-pyrophosphate (PRib-PP) to the 6-oxopurine bases guanine, xanthine, and hypoxanthine to form GMP, XMP, and IMP, respectively. Crystals grown in the absence of substrate or product were used to determine the structure of XPRT at a resolution of 1.8 Angstrom by multiple isomorphous replacement. The core structure of XPRT includes a five-stranded parallel B-sheet surrounded by three or-helices, which is similar to that observed in other known phosphoribosyltransferase (PRTase) structures. The XPRT structure also has several interesting features. A glutamine residue in the purine binding site may be responsible for the altered 6-oxopurine base specificity seen in this enzyme compared to other 6-oxopurine PRTases. Also, we observe both a magnesium ion and a sulfate ion bound at the PRib-PP binding site of XPRT. The sulfate ion interacts with Arg-37 which has a cis-peptide conformation, and the magnesium ion interacts with Asp-89, a highly conserved acidic residue in the PRib-PP binding site motif. The XPRT structure also incorporates a feature which has not been observed in other PRTase structures. The C-terminal 12 residues of XPRT adopt an unusual extended conformation and make interactions with a neighboring subunit. The very last residue, Arg-152, could form part of the active site of a symmetry-related subunit in the XPRT tetramer.

The myosin-I-binding protein Acan125 binds the SH3 domain and belongs to the superfamily of leucine-rich repeat proteins

The SH3 domains of src and other nonreceptor tyrosine kinases have been shown to associate with the motif PXXP, where P and X stand for proline and an unspecified amino acid, but a motif that binds to the SH3 domain of myosin has thus far not been characterized. We previously showed that the SH3 domain of Acanthamoeba myosin-IC interacts with the protein Acan125. We now report that the Acan125 protein sequence contains two tandem consensus PXXP motifs near the C terminus. To test for binding, we expressed a polypeptide, AD3p, which includes 344 residues of native C-terminal sequence and a mutant polypeptide, AD3 Delta 977-994p, which lacks the sequence RPKPVPPPRGAKPAPPPR containing both PXXP motifs. The SH3 domain of Acanthamoeba myosin-IC bound AD3p and not AD3 Delta 977-994p, showing that the PXXP motifs are required for SH3 binding. The sequence of Acan125 is related overall to a protein of unknown function coded by Caenorhabditis elegans gene K07G5.1. The K07G5.1 gene product contains a proline-rich segment similar to the SH3 binding motif found in Acan125. The aligned sequences show considerable conservation of leucines and other hydrophobic residues, including the spacing of these residues, which matches a motif for leucine-rich repeats (LRRs). LRR domains have been demonstrated to be sites for ligand binding. Having an LRR domain and an SH3-binding domain, Acan125 and the C. elegans homologue define a novel family of bifunctional binding proteins.

Impairments of movement kinematics in patients with Huntington's disease: A comparison with and without a concurrent task

This study aimed to quantify the efficiency and smoothness of voluntary movement in Huntington's disease (HD) by the use of a graphics tablet that permits analysis of movement profiles. In particular, we aimed to ascertain whether a concurrent task (digit span) would affect the kinematics of goal-directed movements. Twelve patients with HD and their matched controls performed 12 vertical zig-zag movements, with both left and right hands (with and without the concurrent task), to large or small circular targets over long or short extents. The concurrent task was associated with shorter movement times and reduced right-hand superiority. Patients with HD were overall slower, especially with long strokes, and had similar peak velocities for both small and large targets, so that controls could better accommodate differences in target size. Patients with HD spent more time decelerating, especially with small targets, whereas controls allocated more nearly equal proportions of time to the acceleration and deceleration phases of movement, especially with large targets. Short strokes were generally less force inefficient than were long strokes, especially so for either hand in either group in the absence of the concurrent task, and for the right hand in its presence. With the concurrent task, however, the left hand's behavior changed differentially for the two groups; for patients with HD, it became more force efficient with short strokes and even less efficient with long strokes, whereas for controls, it became more efficient with long strokes. Controls may be able to divert attention away from the inferior left hand, increasing its automaticity, whereas patients with HD, because of disease, may be forced to engage even further online visual control under the demands of a concurrent task. Patients with HD may perhaps become increasingly reliant on terminal visual guidance, which indicates an impairment in constructing and refining an internal representation of the movement necessary for its. effective execution. Basal ganglia dysfunction may impair the ability to use internally generated cues to guide movement.

Synthesis of fructans by partially purified fructosyltransferase activities from Lolium rigidum

Sucrose:sucrose fructosyltransferase (SST) activity was partially purified from whole shoots of Lolium rigidum by a combination of affinity chromatography, gel filtration and anion-exchange chromatography. The SST activity co-eluted with some fructan:fructan fructosyltransferase (FFT) and invertase activities and consequently the partially purified preparation was termed the fructosyltransferase (FT) preparation. The SST-like activity in the FT preparation was purified 214-fold and had an apparent molecular mass of 84 000. The FT preparation contained several peptides with an apparent pI of 4.6-4.7. When assayed with sucrose concentrations up to 600 mM, the FT preparation synthesized 1-kestose at all concentrations, and synthesized 6-kestose at concentrations of 150 mM and greater. The K-m of 1-kestose production was 0.2 M. When the FT preparation was assayed at a concentration of activity approximately half that measured in fresh tissue with 100 mM sucrose, 1-kestose, or 6(G)-kestose as substrates, fructans of degree of polymerization (DP) less than or equal to 5 were synthesized. A partially purified FFT activity, free of SST and invertase activities, which synthesized beta-2,1-glycosidic linked oligofructans of DP less than or equal to 6, was combined in vitro with the FT preparation (FFT-FT preparation) to give a ratio of SST:FFT activities similar to that measured in crude enzyme extracts from L. rigidum. The FFT-FT preparation synthesized oligofructans when assayed with 100 mM concentrations of sucrose, 1-kestose or 6(G)-kestose, but was not able to synthesize fructans of DP greater than or equal to 6 even after extended assays of up to 10 h. The FFT-FT preparation was also assayed with 100 mM sucrose with small amounts of concentrated sucrose added periodically during the assay to maintain the substrate concentration. In this assay, the FFT-FT preparation synthesized fructans up to an apparent DP of 17 or greater. The fructans of DP greater than or equal to 6 synthesized in the assay appeared to form two molecular series containing both beta-2,1- and beta-2,6-glycosidic linked fructosyl residues with terminal or internal glucosyl residues. The apparent rate of SST activity in the assay of the FFT-FT preparation was greater than that measured in a similar assay of the FT preparation alone which did not result in fructans with DP greater than or equal to 6. It was concluded that the FFT-FT preparation, when assayed with a continual supply of sucrose, contained a factor which promoted synthesis of fructans of DP greater than or equal to 6 and synthesis of beta-2,B-glycosidic linkages between fructosyl residues.

Regulation and crystallization of phosphorylated and dephosphorylated forms of truncated dimeric phenylalanine hydroxylase

Phenylalanine hydroxylase is regulated in a complex manner, including activation by phosphorylation. It is normally found as an equilibrium of dimeric and tetrameric species, with the tetramer thought to be the active form. We converted the protein to the dimeric form by deleting the C-terminal 24 residues and show that the truncated protein remains active and regulated by phosphorylation. This indicates that changes in the tetrameric quaternary structure of phenylalanine hydroxylase are not required for enzyme activation. Truncation also facilitates crystallization of both phosphorylated and dephosphorylated forms of the enzyme.

Autopathogenic T helper cell type 1 (Th1) and protective Th2 clones differ in their recognition of the autoantigenic peptide of myelin proteolipid protein

We previously generated a panel of T helper cell 1 (Th1) clones specific for an encephalitogenic peptide of myelin proteolipid protein (PLP) peptide 139-151 (HSLGKWLGHPDKF) that induces experimental autoimmune encephalomyelitis (EAE) upon adoptive transfer. In spite of the differences in their T cell receptor (TCR) gene usage, all these Th1 clones required W144 as the primary and most critical TCR contact residue for the activation. In this study, we determined the TCR contact residues of a panel of Th2/Th0 clones specific for the PLP peptide 139-151 generated either by immunization with the PLP 139-151 peptide with anti-B7-1 antibody or by immunization with an altered peptide Q144. Using alanine-substituted peptide analogues of the native PLP peptide, we show that the Th2 clones have shifted their primary contact residue to the NH2-terminal end of the peptide. These Th2 cells do not show any dependence on the W144, but show a critical requirement for L141/G142 as their major TCR contact residue. Thus, in contrast with the Th1 clones that did not proliferate to A144-substituted peptide, the Th2 clones tolerated a substitution at position 144 and proliferated to A144 peptide. This alternative A144 reactive repertoire appears to have a critical role in the regulation of autoimmune response to PLP 139-151 because preimmunization with A144 to expand the L141/G142-reactive repertoire protects mice from developing EAE induced with the native PLP 139-151 peptide. These data suggest that a balance between two different T cell repertoires specific for same autoantigenic epitope can determine disease phenotype, i.e., resistance or susceptibility to an autoimmune disease.

Conformational analysis of LYS(11-36), a peptide derived from the beta-sheet region of T4 lysozyme, in TFE and SDS

The solution conformation of a peptide LYS(11-36), which corresponds to the beta-sheet region in T4 lysozyme, has been examined in aqueous solution, TFE, and SDS micelles by CD and H-1 NMR spectroscopy. Secondary structure predictions suggest some beta-sheet and turn character in aqueous solution but predict a helical conformation in a more hydrophobic environment. The predictions were supported by the CD and NMR studies which showed the peptide to be relatively unstructured in aqueous solution, although there was some evidence of a beta-turn conformer which was maintained in 200 mM SDS and, to a lesser extent, in 50% TFE. The peptide was significantly helical in the presence of either 50% TFE or 200 mM SDS. TFE and SDS titrations showed that the peptide could form helical, sheet, or extended structure depending on the TFE or SDS concentration. The studies indicate that peptide environment is the determining factor in secondary structure adopted by LYS(11-36).

Structural characterisation of galactoglucomannan secreted by suspension-cultured cells of Nicotiana plumbaginifolia

Galactoglucomannan (GGM) from cultures of Nicotiana plumbaginifolia has Man:Glc:Gal:Ara:Xyl in 1.0:1.1:1.0:0.1:0.04 ratio. Linkage analysis contained 4- and 4,6-Manp, 4-Glcp, terminal Galp and 2-Galp, small amounts and terminal Arap and terminal Xylp, and similar to 0.03 mol acetyl per mol of glucosyl residue. Treatment with alpha- and beta-D-galactosidases showed that the majority of the side-chains were either single Galp-alpha-(1 --> residues or the disaccharide Galp-beta-(1 --> 2)-Galp-alpha-(1 --> linked to O-6 of the 4-Manp residues of the glucomannan backbone. Analysis of the oligosaccharides generated by endo-(1 --> 4)-beta-mannanase digestion confirmed that the GGM comprises a backbone of predominantly alternating --> 4)-D-Manp-beta-(1 --> and --> Lt)-D-Glcp-beta-(1 --> branched at O-6 of 65% of the 4-Manp residues. The major oligosaccharide identified was D-Glcp-beta-(1 --> 4)-[D-Galp-beta-(1 --> 2)-D-Galp-alpha-(1 --> 6)]-D-Manp-beta-(1 --> 4)-D-Glcp-beta-(I --> 4)-[D-Galp-alpha-(1 --> 6)]-D-Manp-beta-(1 --> (27%), and most of the other oligosaccharides produced in significant quantities were based on this structure. (C) 1997 Elsevier Science Ltd.

The structure of versutoxin (delta-atracotoxin-Hv1) provides insights into the binding of site 3 neurotoxins to the voltage-gated sodium channel

Background: Versutoxin (delta-ACTX-Hv1) is the major component of the venom of the Australian Blue Mountains funnel web spider, Hadronyche versuta. delta-ACTX-Hv1 produces potentially fatal neurotoxic symptoms in primates by slowing the inactivation of voltage-gated sodium channels; delta-ACTX-Hv1 is therefore a useful tool for studying sodium channel function. We have determined the three-dimensional structure of delta ACTX-Hv1 as the first step towards understanding the molecular basis of its interaction with these channels. Results: The solution structure of delta-ACTX-Hv1, determined using NMR spectroscopy, comprises a core beta region containing a triple-stranded antiparallel beta sheet, a thumb-like extension protruding from the beta region and a C-terminal 3(10) helix that is appended to the beta domain by virtue of a disulphide bond. The beta region contains a cystine knot motif similar to that seen in other neurotoxic polypeptides. The structure shows homology with mu-agatoxin-l, a spider toxin that also modifies the inactivation kinetics of vertebrate voltage-gated sodium channels. More surprisingly, delta-ACTX-Hv1 shows both sequence and structural homology with gurmarin, a plant polypeptide. This similarity leads us to suggest that the sweet-taste suppression elicited by gurmarin may result from an interaction with one of the downstream ion channels involved in sweet-taste transduction. Conclusions: delta-ACTX-Hv1 shows no structural homology with either sea anemone or alpha-scorpion toxins, both of which also modify the inactivation kinetics of voltage-gated sodium channels by interacting with channel recognition site 3. However, we have shown that delta-ACTX-Hv1 contains charged residues that are topologically related to those implicated in the binding of sea anemone and alpha-scorpion toxins to mammalian voltage-gated sodium channels, suggesting similarities in their mode of interaction with these channels.